The regulation of translational initiation in an in vitro system prepared from cultured human cells (HeLa line) is being investigated, with the aim of identifying specific steps in initiation which may be sensitive to uncharged rRNA. Previous work on this project with intact normal and malignant cells has indicated that in vivo uncharged tRNA may cause a general repression of translational initiation. As part of this work the various partial steps of initiation and their requirements for partially purified initiation factors are being examined. A secondary aim of the project is to develop a means of assaying for mRNA for ribosomal proteins, in poly A-containing mRNA extracted from human cells exposed to various growth stresses. The intent is to determine how the synthesis of these proteins is regulated.